MON-490 Comprehensive Analysis of RCCX Heterogeneity and CAH-X Syndrome in Congenital Adrenal Hyperplasia Using Long-Read Sequencing

Abstract Disclosure: J. Kim: None. J. Park: None. N. Kwak: None. S. Park: None. J. Yoon: None. S. Kim: None. D. Kim: None. S. Hwang: None. G. Kim: None. J. Kim: None. J. Choi: None. H. Yoo: None. Purpose: 21-hydroxylase, encoded by CYP21A2 within the RCCX module, is a multiallelic and highly complex copy number variation locus. Defects in CYP21A2 cause congenital adrenal hyperplasia (CAH). Due to the high degree of homology between active and pseudogenes, the RCCX module is highly susceptible to genetic rearrangements, which can result in a contiguous gene deletion syndrome involving CYP21A2 and the adjacent TNXB gene, known as CAH-X syndrome. This study aimed to analyze the structure of RCCX alleles and identify CAH-X syndrome using long-read sequencing (LRS). Methods: This study included 13 patients with CAH, consisting of 11 with the salt-wasting type and 2 with the simple virilizing type, all of whom had at least one allele deletion of CYP21A2 confirmed by conventional methods. After obtaining informed consent, LRS was performed using the Revio platform from Pacific Biosciences (PacBio, California, USA). The analysis was conducted in a genome assembly format to accurately characterize genetic rearrangements within the RCCX locus. This approach included phasing the RCCX locus for each individual, categorizing CYP21A2 and CYP21A1P genes using pseudogene-specific markers. Additionally, generalized joint hypermobility was assessed to evaluate symptoms and signs of hypermobile Ehlers-Danlos Syndrome (EDS) using Beighton score. Results: In the analysis of 26 genome assemblies obtained from 13 patients, 7 different haplotypes within the RCCX region were identified: 77% (20/26) were bimodular, 12% (3/26) were monomodular, and 12% (3/26) were trimodular. Among the bimodular alleles, 50% (10/20) contained one pseudogene and one CYP21A2 gene, while 20% (4/20) had both a pseudogene and a CYP21A1P/CYP21A2 fusion gene. Additionally, 15% (3/20) exhibited two pseudogenes, and the remaining alleles contained more complex variations. Of the 3 monomodular alleles, two containted a CYP21A1P/CYP21A2 fusion gene, and one contained only a pseudogene. Among the trimodular alleles, two had two pseudogenes and one CYP21A2 gene, while one included two CYP21A1P copies and one CYP21A1P/CYP21A2 fusion gene. Additionally, 4 alleles were identified with TNXA/TNXB fusions: two were classified as CAH-X CH1 alleles, featuring a 120 bp deletion in TNXB exon 35, and two were classified as CAH-X CH2 alleles, characterized by the c.12174C>G variant in TNXB exon 40. The two patients carrying CAH-X CH1 alleles exhibited joint hypermobility (Beighton score 4/9) and easy bruising, consistent with features of CAH-X syndrome. Conclusion: The LRS characterized the variable haplotypes of the RCCX allele and classified the types of CYP21A1P/CYP21A2 and TNXA/TNXB chimeras. Additionally, LRS identified CAH-X syndrome in four patients (31%), including asymptomatic individuals. Presentation: Monday, July 14, 2025